Synergistic blends of antimicrobials useful for controlling microorganisms in industrial processes

ABSTRACT

The present invention provides a method of controlling bacterial contamination using synergistic interactions of antimicrobials. The invention consists of combinations of chlorine dioxide and organic acid whose combined antimicrobial effect is greater than the sum of their individual activities, i.e., synergistic.

This application claims the benefit of U.S. provisional application No.61/791,168, filed Mar. 15, 2013, the entire contents of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The invention relates to synergistic combinations of antimicrobials andmethods of their use for the control of microorganisms in industrialprocesses, materials, or products where their presence is consideredundesirable.

BACKGROUND OF THE INVENTION

It is known that the presence of microorganisms in industrial watersystems may be a significant problem in industrial processes, causingissues with decreased product yields, product quality, and processefficiency.

The physical presence of microbes may causes problems, such as theirgrowth in biofilms on heat exchanging surfaces where they causereductions in heat transfer efficiency. The ability of microbes toconsume a wide variety of materials may cause reductions in yields, forexample, when microbe consuming cellulose cause yield loss in thepaper-making industry. In addition, the production of metabolic productsby contaminating microbes may cause issues, such as their production ofacidic products which may cause product quality issues or contribute tocorrosion issues.

However, in some industries microorganisms are used to produce a numberof fermentation products, such as industrial grade ethanol, distilledspirits, beer, wine, pharmaceuticals and nutraceuticals (foodstuff thatprovides health benefits, such as fortified foods and dietarysupplements), baking industry and industrial chemicals. In theseinstances it is desirable to suppress the growth of unwanted microbesand promote the growth of the wanted ones. In this context the unwantedmicrobes are those which compete for substrate with or produce metabolicproducts that interfere with the growth of the wanted microbes which areproducing the desired end product.

Yeast are commonly used microbes in fermentation processes. One commontype of yeast is Saccharomyces cerevisiae, the species predominantlyused in baking and fermentation. Non-Saccharomyces yeasts, also known asnon-conventional yeasts, are also used to make a number of commercialproducts.

Other microorganisms can also be useful in making fermentation products.For example, cellulosic ethanol production, production of ethanol fromcellulosic biomass, utilizes fungi and bacteria. Examples of thesecellulolytic fungi include Trichoderma reesei and Trichoderma viride.One example of a bacteria used in cellulosic ethanol production isClostridium ljungdahlii.

Most of the yeast used in distilleries and fuel ethanol plants arepurchased from manufacturers of specialty yeasts. The yeast ismanufactured through a propagation process. Propagation involves growinga large quantity of yeast from a small lab culture of yeast. Duringpropagation, the yeast are provided with the oxygen, nitrogen, sugars,proteins, lipids and ions that are necessary or desirable for optimalgrowth through aerobic respiration.

Once at the distillery, the yeast can undergo conditioning. Conditioningis unlike propagation in that it does not involve growing a largequantity from a small lab culture. During conditioning, conditions areprovided to re-hydrate the yeast, bring them out of hibernation andallow for maximum anaerobic growth and reproduction. The objective ofboth propagation and conditioning is to deliver a large volume of yeastto the fermentation tank with high viability, high budding and a lowlevel of infection by other microorganisms.

Following propagation and/or conditioning, the yeast enters thefermentation process. The yeast is combined in an aqueous solution withfermentable sugars. The yeast consumes the sugars, converting them intoaliphatic alcohols, such as ethanol.

The fermentation process begins with the preparation of a fermentablecarbohydrate. In ethanol production, corn is one possible source offermentable carbohydrate. Other carbohydrate sources including cerealgrains and cellulose-starch bearing materials, such as wheat or milo,could also be substituted. Cellulosic biomass such as straw andcornstalks could also be used. Cellulosic ethanol production hasrecently received attention because it uses readily available nonfoodbiomass to form a valuable fuel.

The propagation, conditioning and fermentation processes can be carriedout using batch or continuous methods. The batch process is used forsmall-scale production. Each batch is completed before a new one begins.The continuous fermentation method is used for large-scale productionbecause it produces a continuous supply without restarting every time.

During the propagation, conditioning or fermentation process the mash orthe fermentation mixture can become contaminated with othermicroorganisms, such as spoilage bacteria. These microorganisms competewith the desired species of yeast for fermentable sugars and retard thedesired bio-chemical reaction resulting in a lower product yield. Theycan also produce unwanted chemical by-products, which can cause spoilageof entire fermentation batches.

Producers of ethanol attempt to increase the amount of ethanol producedfrom one bushel of cereal grains (approximately 56 pounds (25.4kilograms)). Contamination by bacteria lowers the efficiency of yeastmaking it difficult to attain or exceed the desired levels of 2.8-2.9gallons of ethanol per bushel (0.42-0.44 liters per kilogram). Reducingthe concentration of bacteria will encourage yeast propagation and/orconditioning and increase yeast efficiency making it possible to attainand exceed these desired levels.

During any of these three processes the yeast can become contaminatedwith undesirable yeast, bacteria or other undesirable microorganisms.This can occur in one of the many vessels used in propagation,conditioning or fermentation. This includes, but is not limited to,propagation tanks, conditioning tanks, starter tanks, fermentationstanks and piping and heat exchangers between these units.

Bacterial contamination reduces the fermentation product yield in threemain ways. First, the sugars that could be available for yeast toproduce alcohol are consumed by the bacteria and diverted from alcoholproduction, reducing yield. Second, the end products of bacterialmetabolism, such as lactic acid and acetic acid, inhibit yeast growthand yeast fermentation/respiration, which results in less efficientyeast production. Finally, the bacteria compete with the yeast fornutrients other than sugar.

After the fermentation system or vessel has become contaminated withbacteria those bacteria can grow much more rapidly than the desiredyeast. The bacteria compete with the yeast for fermentable sugars andretard the desired bio-chemical reaction resulting in a lower productyield. Bacteria also produce unwanted chemical by-products, which cancause spoilage of entire fermentation batches. Removing these bacteriaallows the desired yeast to thrive, which results in higher efficiencyof production.

As little as a one percent decrease in ethanol yield is highlysignificant to the fuel ethanol industry. In larger facilities, such adecrease in efficiency will reduce income from 1 million to 3 milliondollars per year.

Some methods of reducing bacteria during propagation, conditioning andfermentation take advantage of the higher temperature and pH toleranceof yeast over other microorganisms. This is done by applying heat to orlowering the pH of the yeast solution. However, these processes are notentirely effective in retarding bacterial growth. Furthermore, thedesirable yeast, while surviving, are stressed and not as vigorous orhealthy and do not perform as well.

The predominant trend in the ethanol industry is to reduce the pH of themash (feed stock) to less than 4.5 at the start of fermentation.Lowering the pH of the mash reduces the population of some species ofbacteria. However it is much less effective in reducing problematicbacteria, such as lactic-acid producing bacteria or acetic acidproducing bacteria. It also significantly reduces ethanol yield bystressing the yeast used for ethanol production.

Another approach involves washing the yeast with phosphoric acid. Thismethod does not effectively kill bacteria. It can also stress the yeastused for ethanol production, thereby lowering their efficiency.

Yet another method is to use heat or harsh chemicals to sterilizeprocess equipment between batches. It is ineffective at killing bacteriawithin the yeast mixture during production.

Another approach involves the application of chlorine dioxide, anoxidative antimicrobial, generally to the feedstock or recycled watersused in fermentation. Chlorine dioxide is often generated in situ. Veryoften high levels are used to overcome the negating effects of highorganic loads typically seen with oxidative antimicrobials. The chlorinedioxide may be applied at multiple locations in the process, but highlevels in the fermentation tank are avoided since high levels may alsoinhibit yeast. The large volumes of the systems to be treated and thelimited capabilities of current chlorine dioxide generating systemsoften limits the fermentation systems that can be treated with thisapproach or requires the deployment of multiple generators.

In yet another method, antibiotics are added to yeast propagation,conditioning or fermentation batch to neutralize bacteria. Fermentationindustries typically apply antibiotics to conditioning, propagation andfermentation processes. Antibiotic dosage rates range between 0.1 to 3.0mg/L and generally do not exceed 6 mg/L. However, problems exist withusing antibiotics in conditioning, propagation and fermentation.Antibiotics are expensive and can add greatly to the costs oflarge-scale production. Moreover, antibiotics are not effective againstall strains of bacteria, such as antibiotic-resistant strains ofbacteria. Overuse of antibiotics can lead to the creation of additionalvariants of antibiotic-resistant strains of bacteria.

Currently, almost all U.S. biorefining plants utilize an antimicrobialagent and many of them use antibiotics such as virginiamycin. Animportant product of corn biorefining is dried distillers grains for useas animal feed, and the market for antibiotic-free feed grains isgrowing. Distillers grain is the grain residue of the fermentationprocess. Antibiotic residues and establishment of antibiotic-resistantstrains is a global issue. These concerns may lead to future regulatoryaction against the use of antibiotics. It is expected that the FDA willsoon form regulations reducing or eliminating antibiotic use in animalfeed. Canada has similar concerns regarding antibiotics in distillersgrains and most of their production is exported. Europe has alreadybanned the use of antibiotics in ethanol plants where distillers grainsare produced for animal feed. In Brazil, operating antibiotic-free ismandatory in plants producing yeast extract for export. Distiller grainssales account for up to 20% of an ethanol plant earnings. Antibioticconcentration in the byproduct can range from 1-3% by weight, thusnegating this important source of income

In addition, there are other issues to consider when using antibiotics.Mixtures of antibiotics should be frequently balanced and changed inorder to avoid single uses that will lead to antibiotic-resistantstrains. Sometimes the effective amount of antibiotic cannot be added tothe fermentation mixture. For example, utilizing over 2 mg/L ofVirginiamycin will suppress fermentation but over 25 mg/L is required toinhibit grown of Weisella confusa, an emerging problematic bacteriastrain. Overdosing or overuse of antibiotic can stress yeast and impactefficiency or cause regulatory non-compliance.

Industries that employ fermentation for beverages have historicallyapplied hops acid to propagation and fermentation to control unwantedmicrobes that compete with the yeast for nutrients. With the recentexpansion of fuel ethanol, hops acids have been utilized to a minordegree to address unwanted, gram positive microbes. Competition betweenyeasts and unwanted microbes results yield loss of fuel ethanol asunwanted microbes, primarily Lactobacillus and Acetobacter, reduce theefficiency of fermentation. In beverage, competing microbes not onlyreduce efficiency but can alter the aesthetics and taste of the finalproduct.

Organic acid have many applications, including being used as acidifiers,buffers, antioxidants, chelators, synergists, dietary supplements,flavoring agents, preservatives and antimicrobials. The mode of actionof organic acid is that the non-dissociated acids penetrate thebacterial cell wall via passive diffusion and disrupt the normalphysiology of the cell in two ways: The acids dissociate and thereforelower the internal pH, which is normally close to neutral, impairing thefunction of the bacteria. The anionic part of the acid that is unable toleave the cell in its dissociated form accumulates within, disruptingmetabolic functions and increasing osmotic pressure. A drawback to theuse organic acids is the relatively high levels and volumes requiredwhen they are used by themselves.

Since small decreases in ethanol yield are highly significant to thefuel ethanol industry, ethanol producers are constantly looking for waysto increase efficiency. The control of microbes is very significant tomany other industries as well and the predominant strategy is treatmentwith antimicrobials. Antimicrobials are used to eliminate, reduce orotherwise control the number of microbes in aqueous systems. However,the use of most antimicrobials will add cost to operations and productsand thus more effective ways to achieve microbial control are sought. Inaddition, many antimicrobials have deficiencies in either their spectrumof antimicrobial action or operational limitations in their manner ofapplication such as lack of temperature stability or susceptibility toinactivation by environmental or chemical factors. Furthermore, in theinstance of facilities using chlorine dioxide or other in situ generatedantimicrobials, limitations on the volume of antimicrobial able to beproduced may be significant.

Therefore, combinations of antimicrobials may be used, and inparticular, synergistic combinations of antimicrobials are preferred.Synergistic combinations of antimicrobials can deliver an antimicrobialeffect greater than the sum of the individual antimicrobials and thuscan provide an improved cost performance over those combinations whichare merely additive in terms of antimicrobial efficacy. In addition,synergistic combinations of antimicrobials in which one is an in situgenerated antimicrobial may reduce the required volume of antimicrobialand thus increase the maximum size of the system which can be treated.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a platemap of a ternary checkerboard synergy plate, showing anexample of the concentrations of antimicrobials.

FIG. 2 is a graph depicting the bacterial concentrations at time pointsafter antimicrobial addition and at the end of fermentation (64 hours).

FIG. 3 is a graph depicting the average ethanol yield for treatmentsexpressed as grams ethanol per grams of dry corn.

DESCRIPTION OF THE INVENTION

For the purposes of this specification, the meaning of “microorganisms”and “microbes” includes, but is not limited to, bacteria, fungi, algae,protozoans, and viruses. Preferred microbes against which thesecompositions are effective are bacteria, and even more preferred arelactic acid bacteria, acetic acid bacteria, and bacteria which competefor nutrients with yeast in fermentation processes. The microbes can beeither aerobic, anaerobic, or facultative with respect to oxygen use. Itis also understood that the microbes within water or aqueous systems canbe located or suspended within the fluid (eg., planktonic) or localizedon a surface in contact with the aqueous system (eg., biofilms). Thewords and phrases “control”, “microbial control”, “controlling”, and“antimicrobial efficacy” should be broadly construed to include withintheir meaning, without being limited to, inhibiting the growth ofmicrobes, killing microbes, disinfection, preservation, sanitization, orpreventing the re-growth of microbes.

As used herein, ppm is measured as mass per volume, or 1 ppm equals 1 mg(active) per liter. Dosage is defined as the concentration of thecomponent in the system being treated.

As used herein, the term “organic acid” is also referring to its salt.For purposes of the invention the first organic acid is a differentorganic acid then the second organic acid.

The purpose of the present invention is to achieve a significantreduction of the number of contaminating bacteria in industrialprocesses, materials, or products where their presence is consideredundesirable.

The invention provides a synergistic antimicrobial combinationcomprising three parts and methods of using the synergisticantimicrobial combination in the control of microorganisms. Thecomposition comprises chlorine dioxide together with hops acids and anorganic acid. The composition can comprise chlorine dioxide inconjunction with a first organic acid and a second organic acid wherethe first organic acid is different from the second organic acid.Preferred organic acids include citric acid, propionic acid, and benzoicacid or their salts. The preferred first organic acid is citric acid.The acids named refer to themselves or their salts.

It has been discovered that these combinations are synergistic in wateror aqueous systems when used for microbial control, and are alsoeffective in matrices such as those found in biorefining when cornsolids may be present at high levels. Thus, the combined antimicrobialmaterials result in improved antimicrobial efficacy beyond that whichwould be expected based on the sum of their individual antimicrobialefficacies. This unexpectedly observed synergy permits reduced amountsof the antimicrobials to be used to achieve acceptable microbial controlin industrial processes where water is present.

The present invention provides synergistic antimicrobial combinationscomprising chlorine dioxide, hops acid and at least one organic acid andmethods of using the combinations of chlorine dioxide, hops acid, and atleast one organic acid. The invention also provides synergisticantimicrobial combinations comprising chlorine dioxide, at least onefirst organic acid and at least one second organic acid and methods ofusing the combinations of chlorine dioxide, hops acid, and at least oneorganic acid or the combination of chlorine dioxide, at least one firstorganic acid and at least one second organic acid. Preferred organicacids include citric acid, propionic acid, and benzoic acid or theirsalts. The most preferred organic acid is citric acid. The organic acidscan be used in their acid form or their salt form. These combinationsare useful for controlling microorganisms in aqueous systems andproducts. The present invention results in a significant reduction ofthe number of contaminating bacteria in industrial processes, materials,or products where their presence is considered undesirable.

It has been discovered that using the combination of chlorine dioxide,at least one hops acid extract and at least one organic acid or thecombination of chlorine dioxide, at least one first organic acid and atleast one second organic acid provides synergistic microbial control inaqueous systems. Thus, the combination of components result in improvedantimicrobial efficacy beyond that which would be expected based on thesum of their individual antimicrobial efficacies. This unexpectedlyobserved synergy permits reduced amounts of the antimicrobials to beused to achieve acceptable microbial control in industrial processes,such as biorefining, or materials where desired. In instances in whichone antimicrobial is produced in situ such as chlorine dioxide, thereduction in the amount of antimicrobial required allows thecombinations to be used in systems whose volume requirements wouldotherwise be too large to be treated by chlorine dioxide alone.

The composition components may be formulated as a single mixture andadded to the system to be treated. They may also be blended after the insitu generation of the chlorine dioxide and added to the system, or theymay be added sequentially or at different locations in the process. Aperson of ordinary skill in the art can readily determine theappropriate method of addition for each system to be treated.

One non-limiting embodiment of the current method for reducingundesirable microorganism concentration in an aqueous system comprises:

-   -   (a) introducing chlorine dioxide into the system to be treated,    -   (b) introducing a hops acids into the system to be treated, and    -   (c) introducing at least one organic acid into the system to be        treated    -   wherein the chlorine dioxide is at a concentration of at least 1        ppm in the aqueous system to being treated and the concentration        of hops acid is at least 0.5 ppm in the aqueous system to be        treated. Preferred organic acids include citric acid, propionic        acid, and benzoic acid or their salts, most preferred is citric        acid or its salt.

Another non-limiting embodiment of the current method for reducingundesirable microorganism concentration in an aqueous system comprises:

-   -   (a) introducing chlorine dioxide into the system to be treated,    -   (b) introducing at least one first organic acid into the system        to be treated, and    -   (c) introducing at least one second organic acid into the system        to be treated,    -   wherein the chlorine dioxide is at a concentration of at least 1        ppm in the aqueous system to being treated and the total        concentration of the organic acids (sum concentration of the        first organic and the second organic acid) is at least 100 ppm        in the aqueous system to be treated. Preferred organic acids        include citric acid, propionic acid, and benzoic acid or their        salts, most preferred is citric acid or its salt.

The chlorine dioxide used may be generated in situ via a chemicaltransformation of chlorite or chlorate or other substrate, viaelectrochemical generation, or may be provided by stabilizedformulations of chlorine dioxide.

Non-limiting examples of hops acids that can be used in the inventioninclude beta acid compounds, alpha acids, isomerized alpha acids, rhoisomerized alpha acids, tetra isomerized alpha acids, hexa isomerizedalpha acids and hop leaf.

Citric acid is the preferred acid useful in the invention but it canreasonably be expected that other organic acids with a similarantimicrobial mechanism would be useful in the present invention. Thesalts of these acids are also included. Suitable, non-limiting examplesof organic acids useful in the present invention include but are notlimited to citric acid, benzoic acid, propionic acid, tartaric acid,acetic acid, benzenesulfonic acid, oxalic acid, malic acid, salicylicacid, lactic acid, gluconic acid, hydroxyacetic acid and their salts.For purposes of this invention the organic acid is not a hops acid.Preferred organic acids include citric acid, propionic acid, and benzoicacid or their salts. In systems containing at least one first organicacid and at least one second organic acid, citric acid is the preferredfirst organic acid.

The chlorine dioxide can be used in amounts of from 1 ppm to 100 ppm inthe system to be treated. The chlorine dioxide could be used in amountof from 1 ppm to 75 ppm in the aqueous system to be treated or from 1ppm to 50 ppm, or from 1 ppm to 15 ppm, or from 3 ppm to 50 ppm, or from3 ppm to 15 ppm, or from 5 ppm to 20 ppm, or from 5 ppm to 15 ppm.Generally at least 1 ppm or at least 5 ppm or at least 7 ppm of thechlorine dioxide is used. The ratio of the chlorine dioxide to at leastone organic acid can be from 1:1 to 1:15,000, or 1:1 to 1:10,000, orratios of from 1:1 to 1:2000, or ratios of from 1:1 to 1:1000, or ratiosof from 1:4 to 1:10,000, or ratios of from 1:4 to 1:2000, or ratios offrom 1:4 to 1:1000, or from 1:20 to 1:100. The ratios are based on ppmof material in the aqueous system being treated. Hops acids dosages ofat least 0.5 ppm or at least 1 ppm or at least 3 ppm or at least 5 ppmare used. The hops acid dosages can be at least 0.5 ppm and equal to orless than 15 ppm or between 2 ppm and equal to or less than 15 ppm or adosage of from 3 ppm and 10 ppm or between 5 ppm and equal to or lessthan 15 ppm or between 5 ppm and 10 ppm can be used in the invention.The ratio of chlorine dioxide to hops acids can be from 100:1 to 1:10,or from 100:1 to 1:5, or from 75:1 to 1:5, or from 75:1 to 1:2, or from5:1 to 1:5, or 2:1 to 1:2.

One embodiment of the invention comprises citric acid or its salt as theorganic acid in combination with chlorine dioxide and hop acids.Preferably there is at least 1 ppm chlorine dioxide and at least 1 ppmhops acid and at least 50 ppm or 75 ppm or 100 ppm citric acid in thesystem being treated.

One embodiment of the invention comprises propionic acid or its salt asthe organic acid in combination with chlorine dioxide and hop acids.Preferably there is at least 1 ppm chlorine dioxide and at least 1 ppmhops acid and at least 50 ppm or 75 ppm or 100 ppm propionic acid in thesystem being treated.

One embodiment of the invention comprises benzoic acid or its salts asthe organic acid in combination with chlorine dioxide and hop acids.Preferably there is at least 1 ppm chlorine dioxide and at least 1 ppmhops acid and at least 50 ppm or 75 ppm or 100 ppm benzoic acid in thesystem being treated.

In the ternary system comprising chlorine dioxide organic and twoorganic acids, the organic acids are added to the systems to be treatedin the ratios of the first acid to the second acid of from 64:1 up to1:32, or ratios of from 32:1 to 1:32, or ratios of from 32:1 to 1:16, orratios of from 8:1 to 1:32, or ratios of from 8:1 to 1:16 or ratios offrom 8:1 to 1:8. The first acid can be used in amounts of from 12500 ppmdown to 100 ppm in the system to be treated. The first acid could beused in amount of from 6250 down to 100 ppm in the aqueous system to betreated or from 4000 down to 100 ppm or from 4000 down to 200 ppm.Generally at least 100 ppm or at least 200 ppm or at least 300 ppm ofthe first acid is used. The ratio of the first acid to second organicacid can be from 64:1 to 1:32 or from 32:1 to 1:32 or from 8:1 to 1:32.Generally the sum total amount of the two organic acid used in thesystem being treated is less than 20,000 ppm, or less than 15,000 ppm orless than 11,000 ppm. Generally the sum total amount of the two acidsused in the system being treated is at least 50 ppm or at least 75 ppmor at least 100 ppm. The chlorine dioxide could be used in amount offrom 1 ppm to 75 ppm in the aqueous system to be treated or from 1 ppmto 50 ppm or from 1 ppm to 15 ppm or from 3 ppm to 50 ppm or from 3 ppmto 15 ppm of from 3 to 9 ppm. Generally at least 1 ppm or at least 3 ppmor at least 5 ppm or at least 7 ppm of the chlorine dioxide is used inthe aqueous system being treated. The ratio of the chlorine dioxide tothe total amount of the two organic acids can be from 1:1 up 1:15,000 orratios of from 1:1 to 1:10000 or ratios of from 1:1 to 1:2000 or ratiosof from 1:1 to 1:1200 or ratios of from 1:4 to 1:15,000 or ratios offrom 1:4 to 1:10000 or ratios of from 1:4 to 1:2000 or ratios of from1:4 to 1:1000 or ratios of from 1:20 to 1:100. The first organic acidand the second organic acid are not the same organic acid nor itsrespective salt. The preferred organic acid for the first organic acidis citric acid or its salt.

Examples of water and aqueous systems in which the compositions areuseful are biorefining, cooling water, boiler water, pulp and paper millwater, oil and gas field injection water and produced water, oil and gaspipelines and storage systems, fuel, ballast water, wastewater,pasteurizers, other industrial process water, metalworking fluids,latex, polymers, paint, coatings, adhesives, inks, personal care andhousehold products, reverse osmosis systems, electrochemical depositionsystems, fluids used in mineral extraction, mineral slurries,agricultural processing, biorefining waters, and systems that use them.In addition, the compositions may be used in other areas where microbialcontamination of water and aqueous systems is required. Suchapplications include washing fruits and vegetables during processing,and the treatment of waters used in food processing such as their use intunnel pasteurizers.

The composition components can be combined and then added to the systemto be treated. They may also be added sequentially, from a single dosingpoint, or from separate dosing points. The components of the compositioncan be added to the water or aqueous system separately or blended priorto addition. A person of ordinary skill in the art can readily determinethe appropriate method of addition. The composition can be added to thewater or aqueous system with other additives such as, but notnecessarily restricted to, surfactants, scale and corrosion controlcompounds, ionic or non-ionic polymers, pH control agents, and otheradditives used for altering or modifying the chemistry of the water oraqueous system. In addition, the compositions' may be used in water andaqueous systems which contain antimicrobial agents other than thoselisted herein as synergistic

The pH of the aqueous system to be treated is generally from 3 to 11, orfrom 3 to 7, or from 4 to 9, or from 4 to 8, or from 4 to 6.5, or from4.5 to 6. In general, the organic acids work best in systems where thepH of the system is less than at least one of the pKa values of the acidor its salt.

In some non-limiting embodiments, the synergistic aqueous solution iscomprised of chlorine dioxide and hops acid extracts and citric acid orits salt where the hops acid to organic acid is in ratios of 1:10 to1:6500, or 1:25 to 1:6400, or 1:25 to 1:1600, or from 1:25 to 1:500 orfrom 1:25 to 1:100

In some non-limiting embodiments, the synergistic aqueous solution iscomprised of chlorine dioxide and hops acid extracts and propionic acidor its salt where the hops acid to organic acid is in ratios of 1:12.5to 1:800, preferably 1:12.5 to 1:400, preferably from 1:12.5 to 1:50 or1:10 to 1:6500, or 1:25 to 1:6400, or 1:25 to 1:1600, or from 1:25 to1:500.

In one embodiment the synergistic aqueous solution is comprised ofchlorine dioxide and hops acid extracts and the organic acid is citricacid or its salt. Citric acid could be used in an amount of 1000 ppmdown to 50 ppm or from 800 down to 75 ppm or from 600 down to 100 ppm inthe aqueous system to be treated. Generally at least 50 ppm or at least75 ppm or at least 100 ppm of citric acid is used in the aqueous systemto be treated. The amount of chlorine dioxide used in the aqueous systemto be treated is from 1 ppm to 50 ppm, or from 1 ppm to 15 ppm or from 1ppm to 10 ppm or from 5 ppm to 10 ppm, and the more preferredcomposition has 5 to 10 ppm chlorine dioxide or from 3 to 9 ppm chlorinedioxide.

The invention provides synergistic antimicrobial combinations andmethods of using them in the control of microorganisms, for example inindustrial fermentations producing ethanol or other chemicals.

When used in a fermentation system the combination of chlorine dioxideand hops acid extract and organic acid, preferably citric acid, can beadded in various locations in the fermentation system, including theslurry tank(s), cookers, mash coolers, propagators and fermentationtanks. One skilled in the art may also determine other addition points.

In fermentation systems using the present method, the concentrations ofbacteria and other undesirable microorganisms can be reduced whilepropagation and/or conditioning of desirable microorganisms isencouraged. It has been discovered that chlorine dioxide in combinationwith at least one hops acid extract and at least one organic acid,preferably citric acid, or chlorine dioxide in combination with at leastone first organic acid, preferably citric acid and at least on secondorganic acid is effective at reducing the concentration of undesirablebacteria and other undesirable microorganisms while simultaneouslyencouraging propagation and/or conditioning of desirable microorganisms.The combination of these products provides a synergistic, antimicrobialtreatment without the use of antibiotics.

One non-limiting embodiment of the current method for reducingundesirable microorganism concentration, promoting desirablemicroorganism propagation, and increasing desirable microorganismefficiency in an aqueous system comprises:

-   -   (a) introducing a fermentable carbohydrate to an aqueous system,    -   (b) introducing at least one yeast or desirable microorganism to        the aqueous system, and    -   (c) introducing chlorine dioxide and at least one hops acid        extract and at least one organic acid to the aqueous system.        The chlorine dioxide and the hops acid extract and the organic        acid can be introduced into the aqueous system as a blend, or        individually, or any two as a blend and the third individually.        Preferably the organic acid is citric acid.

Another non-limiting embodiment of the current method for reducingundesirable microorganism concentration, promoting yeast propagation,and increasing yeast efficiency in an aqueous system comprises:

-   -   (a) introducing a quantity of fermentable carbohydrate to an        aqueous system,    -   (b) introducing a quantity of yeast to the aqueous system, and    -   (c) introducing chlorine dioxide and at least one hops acid        extract and at least one organic acid into the aqueous system.        The chlorine dioxide and the hops acid extract and the organic        acid can be introduced into the aqueous system as a blend, or        individually, or any two as a blend and the third individually.        Preferably the organic acid is citric acid.

One non-limiting embodiment of the current method for reducingundesirable microorganism concentration, promoting desirablemicroorganism propagation, and increasing desirable microorganismefficiency in an aqueous system comprises:

-   -   (a) introducing a fermentable carbohydrate to an aqueous system,    -   (b) introducing at least one yeast or desirable microorganism to        the aqueous system, and    -   (c) introducing chlorine dioxide and at least one first organic        acid, preferably citric acid, and at least one second organic        acid that is not citric acid to the aqueous system, wherein the        concentration of chlorine dioxide in the system being treated is        at least 1 ppm or at least 5 ppm or at least 10 ppm and the sum        concentration of organic acids is at least 50 ppm or at least 75        ppm.        The chlorine dioxide and the at least one first organic acid and        the at least one second organic acid can be introduced into the        aqueous system as a blend, or individually, or any two as a        blend and the third individually. Citric acid sit he preferred        first organic acid

Another non-limiting embodiment of the current method for reducingundesirable microorganism concentration, promoting yeast propagation,and increasing yeast efficiency in an aqueous system comprises:

-   -   (a) introducing a quantity of fermentable carbohydrate to an        aqueous system,    -   (b) introducing a quantity of yeast to the aqueous system, and    -   (c) introducing chlorine dioxide and at least one first organic        acid, preferably citric acid, and at least one second organic        acid to the aqueous system, wherein the concentration of        chlorine dioxide in the system being treated is at least 1 ppm        or at least 5 ppm or at least 10 ppm and the sum concentration        of organic acids is at least 50 ppm or 75 ppm.        The chlorine dioxide and the at least one first organic acid and        the at least one second organic acid can be introduced into the        aqueous system as a blend, or individually, or any two as a        blend and the third individually. Citric acid sit he preferred        first organic acid.

The steps of the method can be performed sequentially or in a differentorder. The components of the antimicrobial system can be brought intocontact with the yeast or with the fermentation carbohydrate; or theyeast and the fermentable carbohydrate can be combined and then thecomponents of the antimicrobial system be introduced into thecombination of yeast and carbohydrate. The components of theantimicrobial system can be combined together and then added to theaqueous system or they can be added separately to the aqueous system.The aqueous system can be in a continuous process or may be a tank inthe case of a batch process.

In the foregoing method, the “undesirable” microorganisms intended to bereduced are those that compete for nutrients with the desirablemicroorganisms that promote the desired fermentation processes. Unwantedor undesirable microorganisms in fermentation include the lactic acidproducing bacteria (LAB) and the acetic acid producing bacteria of whichLactobacillus and Acetobacter are prominent representatives. Any microbethat competes for the fermentable substrate, denying it to the intendedfermenting organism and thus reducing yields can be consideredundesirable. In this regard, the chlorine dioxide, organic acid and hopsacid extract employed in the present method preferably do notdetrimentally affect the growth and viability of desirable,fermentation-promoting microorganisms, but do eliminate or suppress thegrowth of undesirable microorganisms that interfere with thefermentation process. Moreover, the elimination or suppression ofundesirable microorganisms has a favorable effect on the growth andviability of desirable microorganisms.

The chlorine dioxide in conjunction with at least one organic acid,preferably citric acid and hops acids can also be used in the treatmentof water used to wash fruits and vegetables. Although chlorine dioxideis used in some cases by itself to wash fruits and vegetables, thepresence of high organic matter loads often requires high concentrationsof chlorine dioxide to be efficacious. Generally the fruit andvegetables are washed by spraying or submerging the fruit or vegetablesin an aqueous solution of the antimicrobials, where the concentrationsof the antimicrobials are those described above. The synergisticcombination of chlorine dioxide, at least one organic acid, preferablycitric acid, and hops acids means that a greater antimicrobial effectcan be achieved with reduced antimicrobial levels. Another applicationof chlorine dioxide, a at least one organic acid, preferably citricacid, and hops acids would be in the production of water used to prepareprocessed food or drinks, or in food hygiene applications like themaintenance of wash water in tunnel pasteurizers. Generally, chlorinedioxide in conjunction with at least one organic acid, preferably citricacid, and hops acids can be used for any application in which thebreakdown of the antimicrobial agents produces only salt, water, and afood additive is a desirable result.

The production of fuel ethanol by yeast fermentation is used as anexample of where the present invention can be used. Other fermentationproducts which could employ the combination of the chlorine dioxide inconjunction with the two organic acids or combination of the chlorinedioxide in conjunction with the organic acid, preferably citric acid,and hops acid could include distilled spirits, beer, wine,pharmaceuticals, pharmaceutical intermediates, baking products,nutraceuticals (foodstuff that provides health benefits, such asfortified foods and dietary supplements), nutraceutical intermediates,industrial chemical feedstocks, and enzymes. The current method couldalso be utilized to treat yeast used in the baking industry.

Saccharomyces yeasts are one type of useful yeast such as Saccharomycescerevisiae. Non-Saccharomyces yeasts can also be used in the invention.Yeast are not the only beneficial microorganisms used in fermentation.Additional desirable fermenting microorganisms could also be used andbenefited by the invention such as the fungi and bacteria typically usedin cellulosic ethanol production. Some non-limiting examples ofdesirable fermenting microorganisms include, but are not limited to,Trichoderma reesei, Trichoderma viride, and Clostridium ljungdahlii.

The components of the antimicrobial system (chlorine dioxide inconjunction with the hops acid extract and at least one organic acid,preferably citric acid, or chlorine dioxide in conjunction with at leastone first organic acid, preferably citric acid, and at least one secondorganic acid) can be added at various points in the propagation,conditioning and/or fermentation processes. The components of theantimicrobial system can be added to cook vessels, fermentation tanks,propagation tanks, conditioning tanks, starter tanks or duringliquefaction. The components of the antimicrobial system can also beadded directly to the corn mash. The components of the antimicrobialsystem can also be added to the interstage heat exchange system or heatexchangers. The components of the antimicrobial system can also be addedto the piping between these units or heat exchangers.

The components of the antimicrobial system can be added directly intothe fermentation mixture. This can be done by adding the components ofthe antimicrobial system in conjunction with the yeast or otherdesirable microorganism and fermentable carbohydrate, for example duringthe SSF (Simultaneous saccharification and fermentation) stage. Thedosage is the concentration of the component in the aqueous system beingtreated. The chlorine dioxide dosages of between 1 and 100 ppm or 1 to75 ppm or 1 to 50 ppm and the hops acid extract dosages of between 0.5and 20 ppm or 0.5 to 15 ppm, or from 1 to 10 ppm and the organic acid,preferably citric acid, dosage of between 75 and 1,000 can be addeddirectly into the fermentation mixture. For the antimicrobial systemwith chlorine dioxide and two organic acids, the chlorine dioxide dosagewould be between 1 and 100 ppm or 1 to 75 ppm or 1 to 50 ppm and the sumtotal of the two organic acids would be at least 50 ppm up to 2000 ppm,preferably citric acids is one of the two acids.

The chlorine dioxide in conjunction with the hops acid extract and atleast one organic acid, preferably citric acid, can also be added to themash prior to the fermentation process. The chlorine dioxide dosages ofbetween 1 and 100 ppm or 1 to 75 ppm or 1 to 50 ppm and the hops acidextract dosages of between 0.5 and 20 ppm or 0.5 to 15 ppm, or from 1 to10 ppm and the organic acid dosage of between 75 and 1,000 can be addeddirectly into the fermentation mixture. The chlorine dioxide incombination with at least one first organic acid, preferably citricacid, and at least on second organic acid can also be added to the mashprior to the fermentation process. For the antimicrobial system withchlorine dioxide and two organic acids, the chlorine dioxide dosagewould be between 1 and 100 ppm or 1 to 75 ppm or 1 to 50 ppm and the sumtotal of the two organic acids would be at least 50 ppm up to 2000 ppm

The chlorine dioxide in conjunction with the hops acid extract and atleast one organic acid, or chlorine dioxide in conjunction with the atleast one first organic acid extract and at least one second organicacid can also be added during propagation and/or conditioning. Forexample the chlorine dioxide in conjunction with the hops acid extractand organic acid can be added to the yeast slurry before SSF replacingan acid washing step.

The antimicrobial systems of the present invention, namely chlorinedioxide in conjunction with hops acid and at least one organic acid orchlorine dioxide in combination with at least one first organic acid andat least on second organic acid, can be used to achieve improved resultsin the production of cellulosic ethanol. Cellulosic ethanol is a type ofethanol that is produced from cellulose, as opposed to the sugars andstarches used in producing carbohydrate based ethanol. Cellulose ispresent in non-traditional biomass sources such as switch grass, cornstover and forestry. This type of ethanol production is particularlyattractive because of the large availability of cellulose sources.Cellulosic ethanol, by the very nature of the raw material, introduceshigher levels of contaminants and competing microorganism into thefermentation process. The antimicrobial systems of the present inventioncan be used in cellulosic ethanol production to control undesirablemicroorganisms. The chlorine dioxide dosages of between 1 and 100 ppm or1 to 75 ppm or 1 to 50 ppm and the hops acid extract dosages of between0.5 and 20 ppm or 0.5 to 15 ppm, or from 1 to 10 ppm and the organicacid dosage of between 75 and 1,000 can be can be used in the productionof cellulosic ethanol. The chlorine dioxide in combination with at leastone first organic acid, preferably citric acid, and at least on secondorganic acid can also be can be used in production of cellulosic ethanolwhere the chlorine dioxide dosage is between 1 and 100 ppm or 1 to 75ppm or 1 to 50 ppm and the sum total of the two organic acids would beat least 50 ppm up to 2000 ppm.

There are two primary processes of producing alcohol from cellulose. Oneprocess is a hydrolysis process that utilizes fungi, as for exampleTrichoderma reesei and/or Trichoderma viride. The other is agasification process using a bacteria such as Clostridium ljungdahlii.The antimicrobial systems of the present invention can be utilized ineither process.

In the hydrolysis process the cellulose chains are broken down into fivecarbon and six carbon sugars before the fermentation process. This iseither done chemically or enzymatically.

In the chemical hydrolysis method the cellulose can be treated withdilute acid at high temperature and pressure or concentrated acid atlower temperature and atmospheric pressure. In the chemical hydrolysisprocess the cellulose reacts with the acid and water to form individualsugar molecules. These sugar molecules are then neutralized and yeastfermentation is used to produce ethanol. The antimicrobial systems ofthe present invention can be used during the yeast fermentation portionof this method.

Enzymatic hydrolysis can be carried out using two methods. The first isknown as direct microbial conversion (DMC). The DMC method uses a singlemicroorganism to convert the cellulosic biomass to ethanol. The ethanoland required enzymes are produced by the same microorganism. Theantimicrobial systems of the present invention can be used during thepropagation/conditioning or fermentation steps with this specializedorganism.

The second method is known as the enzymatic hydrolysis method. In thismethod cellulose chains are broken down using cellulase enzymes. Theseenzymes are typically present in the stomachs of ruminants, such as cowsand sheep, to break down the cellulose that they eat. The enzymaticmethod is typically carried out in four or five stages. The cellulose ispretreated to make the raw material, such as wood or straw, moreamenable to hydrolysis. Next the cellulase enzymes are used to break thecellulose molecules into fermentable sugars. Following hydrolysis, thesugars are separated from residual materials and added to the yeast. Thehydrolyzate sugars are fermented to ethanol using yeast. Finally, theethanol is recovered by distillation. Alternatively, the hydrolysis andfermentation can be carried out together by using special bacteria orfungi that accomplish both processes. When both steps are carried outtogether the process is called sequential hydrolysis and fermentation(SHF).

The antimicrobial systems of the present invention can be introduced formicrobiological efficacy at various points in the enzymatic method ofhydrolysis. The antimicrobial systems of the present invention can beused in the production, manufacture and fermentation of cellulaseenzymes made by Trichoderma and other fungi strains. The antimicrobialsystems of the present invention acid can be added in the cellulosicsimultaneous saccharification and fermentation phase (SSF). Theantimicrobial systems of the present invention can be introduced in thesequential hydrolysis and fermentation (SHF) phase. They could also beintroduced at a point before, during or after the fermentation bycellulolytic fungi that create the cellulase enzymes. Alternatively theantimicrobial systems of the present invention can be added during theyeast fermentation phase, as discussed above.

The gasification process does not break the cellulose chain into sugarmolecules. First, the carbon in the cellulose is converted to carbonmonoxide, carbon dioxide and hydrogen in a partial combustion reaction.Then, the carbon monoxide, carbon dioxide and hydrogen are fed into aspecial fermenter that uses a microorganism such as Clostridiumljungdahlii that is capable of consuming the carbon monoxide, carbondioxide and hydrogen to produce ethanol and water. Finally, the ethanolis separated from the water in a distillation step. The antimicrobialsystems of the present invention can be used as an antimicrobial agentin the fermentation step involving microorganisms such as Clostridiumljungdahlii that are capable of consuming carbon monoxide, carbondioxide and hydrogen to produce ethanol and water.

In one non-limiting embodiment, chlorine dioxide, hops acids and atleast one organic acid are combined in a tank and diluted to apredetermined concentration at a predetermined ratio. In the tank,chlorine dioxide, hops acid, preferably as isomerized alpha extract, andorganic acid, preferably citric acid, are dissolved in water to form achlorine dioxide/hops acids/organic acid blend. The concentration of thechlorine dioxide solution, the hops acid extract solution and theorganic acid solution in the batch tank can vary across a wide range.The blended chlorine dioxide/hops acid extract/organic acid solution isthen exhausted from the batch tank through an outlet at a specifieddosage rate to create a solution of the desired concentration.

In one non-limiting embodiment, chlorine dioxide, at least one firstorganic acid and at least second organic acid are combined in a tank anddiluted to a predetermined concentration at a predetermined ratio. Inthe tank, chlorine dioxide, at least one first organic acid preferablycitric acid, and at least second organic acid, preferably propionic acidor benzoic acid, or their salts, are dissolved in water to form achlorine dioxide/first organic acid/second organic acid blend. Theconcentrations of the chlorine dioxide and the organic acids in solutionin the batch tank can vary across a wide range. The blended chlorinedioxide/hops organic acids solution is then exhausted from the batchtank through an outlet at a specified dosage rate to create a solutionof the desired concentration.

EXAMPLES

The synergy indices reported in the following examples use the followingformula, which is a modification of the formula reported in F. C. Kull,P. C. Eisman, H. D. Sylwestrowka, and R. L. Mayer, Applied Microbiology9:538-541, 1961.

Synergy Index=Qa/QA+Qb/QB+Qc/QC

-   -   where Qa is the concentration of Antimicrobial A required to        achieve complete inhibition of growth of the test microbe when        used in combination with Antimicrobials B and C;    -   QA is the concentration of Antimicrobial A required to achieve        complete inhibition of growth of the test microbe when used        alone;    -   Qb is the concentration of Antimicrobial B required to achieve        complete inhibition of growth of the test microbe when used in        combination with Antimicrobials A and C;    -   QB is the concentration of Antimicrobial B required to achieve        complete inhibition of growth of the test microbe when used        alone;    -   Qc is the concentration of Antimicrobial C required to achieve        complete inhibition of growth of the test microbe when used in        combination with Antimicrobials A and B;    -   QC is the concentration of Antimicrobial C required to achieve        complete inhibition of growth of the test microbe when used        alone.

A synergy index (SI) of 1 indicates the interactions among theantimicrobials is merely additive, a SI of greater than one indicatesthe antimicrobials are antagonistic with each other, and a SI of lessthan 1 indicates the antimicrobials interact in a synergistic manner.

While there are various methods known to individuals skilled in the artfor measuring levels of antimicrobial activity, in the followingexamples the endpoint used is known as the Minimal InhibitoryConcentration, or MIC. This is the lowest concentration of a substanceor substances which can achieve complete inhibition of growth.

In order to determine the Minimal Inhibitory Concentration, a two-folddilution series of the antimicrobial is constructed with the dilutionsbeing made in growth media. The dilutions are made in a 96 wellmicroplate such that each well has a final volume of 280 μl of media andantimicrobial. The first well has, for example, a concentration of 1000ppm antimicrobial, the second 500 ppm, the third 250 ppm, and so forth,with the 12^(th) and final well in the row having no antimicrobial atall and serving as a positive growth control. After the dilution seriesis constructed the wells receive an inoculum of microbe suspended ingrowth media such that the final concentration of microbes in the wellis ˜5×10⁵ cfu/ml. In these examples the test microbe used isLactobacillus plantarum. The cultures are incubated at an appropriatetemperature for 18-24 hours, and the wells scored as positive ornegative for growth based on a visual examination for turbid wells. Thelowest concentration of antimicrobial which completely inhibits growth(e.g. a clear well) is designated the Minimal Inhibitory Concentration.

In order to determine whether the interaction between threeantimicrobials is additive, antagonistic, or synergistic against atarget microbe a modification of the MIC method known as the“checkerboard” method is employed using 96 well microplates. A“checkerboard” synergy plate for two antimicrobials uses an 8×8two-dimensional grid on a single microplate. The ternary synergy methoduses a 8×8×8 three-dimensional grid using a stack of 8 microplates. Toconstruct a checkerboard plate, Antimicrobial A is deployed via thegrowth media. The antimicrobial is dissolved into the growth media anddispensed onto the eight plates; thus each plate has a singleconcentration of Antimicrobial A. There is a total of 8 plates eachcontaining a different concentration of Antimicrobial A. The secondantimicrobial (Antimicrobial B) is deployed using the two-fold serialdilution method used to construct an MIC plate, where each of the eightrows (A-H) is an identical dilution series of decreasing concentrationswhich terminates after the eighth column. The third antimicrobial(Antimicrobial C) is deployed by adding identical volumes of a singleantimicrobial concentration to each column (1-8), with each columngetting a different concentration. Thus, Column 1 receives a volume ofmedia plus Antimicrobial C at 1000 μM, column 2 receives a volume ofmedia plus antimicrobial at 500 μM, etc. The result is each well of the8×8 well grid has a different combination of antimicrobialconcentrations, yielding 64 different combinations of Antimicrobial Band C in total, with Antimicrobial A being held constant. Each of theeight plates has identical grids of Antimicrobials B and C but withdifferent concentrations of Antimicrobial A, yielding a total of 512different combinations of Antimicrobials A plus B plus C. This iseffectively an 8×8×8 grid. The 9^(th) and 10^(th) columns of each platereceive no antimicrobial at all, just media, and serve as positive andnegative growth controls, respectively. After the checkerboardmicroplate is constructed, it is inoculated with Lactobacillusplantarum, incubated at 37° C., and scored as described for the MICmethod. FIG. 1 depicts the checkerboard microplate.

Example 1 Synergy of Chlorine Dioxide with Hops Acids and Citric Acid

Minimal inhibitory concentrations were determined for chlorine dioxide,hops acids and citric acid at pH 5 using the protocol described abovewith Lactobacillus plantarum as the test microbe. Ternary synergy plateswere constructed as described, the wells inoculated to a finalconcentration of ˜5×10⁵ cfu/ml, incubated for 18-24 hours, and thenscored visually for growth/no growth. Synergy indices were calculatedaccording to the modified formula described by Kull et al. This exampledemonstrates that the effect of combining chlorine dioxide, hops acidsand citric acid greater than the effect of any of the antimicrobialsalone. The amount of chlorine dioxide needed to inhibit bacterial growthis reduced from 54 ppm to 2.75-40 ppm. The concentration of hops acidsdrops from 5 ppm to a range of 0.625-2.5 ppm and citric acid goes from6,250 ppm to 78-1250 ppm.

TABLE 1 Used Alone Used in Combination ClO2 Hops Citric Hops MIC AcidMIC Acid MIC ClO2 MIC Acid MIC Citric Acid ClO2:Hops (QA) (QB) (QC) (Qa)(Qb) MIC (Qc) Acid:Citric ppm ppm ppm ppm ppm ppm Acid Ratio SI 54 56250 35 1.25 78 28:1:62.4 0.91 54 5 6250 35 0.625 78 56:1:124.8 0.79 545 6250 18.8 2.5 156 7.52:1:62.4 0.87 54 5 6250 37.5 1.25 156 30:1:124.80.97 54 5 6250 37.5 0.625 156 60:1:249.6 0.84 54 5 6250 20 2.5 3138:1:125.2 0.92 54 5 6250 40 0.625 313 64:1:500.8 0.92 54 5 6250 21.252.5 625 8.5:1:250 0.99 54 5 6250 2.75 2.5 1250 1.1:1:500 0.75

Example 2 Fermentation Lab Data

Evaluations were conducted at the National Corn-to-Ethanol ResearchCenter, utilizing chlorine dioxide, hops acid extracts and citric acid.The samples tested and their concentrations can be found in FIG. 2 andTable 2. The tests were conducted to evaluate the effects of ternaryantimicrobials on ethanol production in corn mash produced underconditions that are similar to those used in the fuel ethanol industry.Two specific effects were investigated: (1) the ability ofantimicrobials to affect ethanol yield and sugar conversion infermentations that are contaminated by lactic acid bacteria, and (2) theability of antimicrobials to control bacterial infections compared tocontrol bacteria-free fermentations. Three 160-gram slurries of cornflour, water and enzyme (30% w/w dry solids) were made for eachtreatment and control (inoculated and uninoculated). The slurries wereincubated for 90 minutes at 83° C., cooled to 40° C., and theninoculated with L. plantarum. Next, the slurries were dosed withantimicrobial. The facility dosed chlorine dioxide, hops acid extractsand citric acid to 250-mL Erlenmeyer flasks and samples were collectedat 15, 30 and 60 minutes post antimicrobial addition. After the 3time-point samples were collected, the pH of the mash was adjusted to<5.2 by addition of 300 μl of 5-N sulfuric acid. All enzymes, nutrients,and other amendments added to the fermentation flasks were freshlyprepared before use. Urea was added as a sterile 0.2-g/ml solution to afinal concentration of 500 ppm (w/w) based on the nitrogen content ofthe urea (w/w, based on the total mass of mash). The glucoamylase enzyme(Spirizyme Excel, Novozymes) was prepared as a 0.25-g/ml solution andadded at a dose of 0.066% (w/w, based on the wet weight of corn).Sterile water was added to equalize the total solids content of eachtreatment. All fermentation flasks were inoculated with a 0.2-g/mlsuspension of yeast (Saccharomyces cerevisiae). This suspension wasincubated and mixed for 30 minutes at 40° C. before inoculation into thefermentation flasks. Each fermentation flask was inoculated with 170 μlof the yeast suspension to attain an initial concentration of 1×10⁷yeast cells/ml. The mass of each flask was recorded after all additionswere made, then sanitized fermentation traps were inserted into eachflask and they were weighed again. The flasks were incubated at 32° C.with shaking at 170 rpm in an incubator/shaker for a total of 64 hours.Fermentation progress was monitored by weighing the fermentation flasksperiodically during the 3-day incubation (at 0, 17.5, 22.5, 42.5, 48,and 64 hrs after inoculation with yeast). The concentrations ofsubstrates (glucose, DP2, DP3, and DP4+, where “DPx” represent glucoseoligomers with “x” subunits) and products (ethanol, glycerol, lacticacid, and acetic acid) were measured by HPLC at the end of fermentation.Samples were prepared for HPLC by centrifugation to remove large solids,followed by filtration through 0.45-μm syringe filters, andacidification to pH of approximately 2 by addition of sulfuric acid to afinal concentration of 0.01 N. The final pH, concentrations of total drysolids and dissolved dry solids, and the density of the beer filtratewere measured after incubation for 64 hours. Samples from each flaskwere plated for bacterial colony counts.

TABLE 2 5 ppm ClO2/5 ppm hops/200 Time Control ppm Citric acid (hours)(CFU/mL) (CFU/mL) 0.25 1,200,000 1,220,000 0.5 1,340,000 1,700,000 110,300,000 3,000,000 64 55,600 12,800

This example shows that during fermentation, 5 ppm of chlorine dioxidecombined with 5 ppm of hops acids combined with 200 ppm of citric acidis effective in reducing bacteria, which was unexpectedly low afterseeing the laboratory MIC and synergy data.

FIG. 3 and Table 3 show the average ethanol yields of the uninfectedcontrol and the three samples after fermentation. No significantdifferences were observed in the average ethanol yields among alltreatments (P=0.769), using ANOVA. In FIG. 3 and table 3 the datarepresent the average of three independent replicate fermentationflasks.

TABLE 3 Ethanol Yield (g ethanol/g Sample dry corn) Infection freecontrol 0.388 5 ppm ClO2/5 ppm Hops/200 ppm Citric acid 0.377

1. A aqueous composition comprising: a) chlorine dioxide, b) hops acids,and c) at least one organic acid or their salts, wherein theconcentration of chloride dioxide is at least 1 ppm; wherein theconcentration of hops acid is at least 0.5 ppm; and wherein theconcentration of the at least one organic acid is at least 50 ppm.
 2. Anaqueous composition comprising: (a) chlorine dioxide, and (b) at leastone first organic acid or its salt, and (c) at least one second organicacids or its salt, wherein the concentration of chloride dioxide is atleast 1 ppm; and wherein the sum concentration of the first organic acidand the second organic acid is at least 50 ppm.
 3. A composition ofclaim 1 wherein the at least one hops acid is selected from the groupconsisting of beta acid compounds, alpha acids, isomerized alpha acids,rho isomerized alpha acids, tetra isomerized alpha acids, hexaisomerized alpha acids and hop leaf or combination thereof.
 4. Thecomposition of claim 1 wherein the at least one organic acid is selectedfrom the group consisting of citric acid, propionic acid, benzoic acid,or their salts and combinations thereof.
 5. The composition of claim 1wherein the at least one organic acid comprises citric acid or its salt.6. The composition of claim 1 wherein the ratio of chlorine dioxide toorganic acid is from 1:1 to 1:15,000.
 7. The composition of claim 2wherein the ratio in ppm of chlorine dioxide to the sum of the firstorganic acid and the second organic acid is from 1:1 to 1:15,000.
 8. Thecomposition of claim 1 wherein the concentration of chloride dioxide isat least 1 ppm to 50 ppm, and wherein concentration of the hops acid isat least 0.5 ppm to 20 ppm, and wherein the concentration of the atleast one organic acid is at least 50 ppm to 5000 ppm.
 9. Thecomposition of claim 2, wherein the at least one first organic acid iscitric acid and the at least second organic acid is selected from thegroup consisting of propionic acid, benzoic acid, or their salts andcombinations thereof.
 10. A method of controlling undesirablemicroorganism concentration in an aqueous system, the method comprisingthe steps of: (a) introducing chlorine dioxide into an aqueous system,(b) introducing an organic acid into the aqueous system, (c) introducingan hops acids into the aqueous system, wherein the organic acid isselected from the group consisting of citric acid, propionic acid,benzoic acid, and their salts and wherein the concentration of chlorinedioxide is at least 1 ppm in the aqueous system being treated.
 11. Themethod of claim 10 wherein the aqueous system is a fermentation process,and the method further comprising the steps of: (x) introducing afermentable carbohydrate to an aqueous solution; (y) introducing atleast one yeast to said solution.
 12. The method of claim 10 wherein theconcentration of chloride dioxide is at least 10 ppm in the aqueoussystem being treated.
 13. The method of claim 10 wherein theconcentration of chloride dioxide is from 1 ppm to 50 ppm in the aqueoussystem being treated.
 14. The method of claim 10 wherein the at leastone organic acid comprises citric acid or its salt, and the ratio ofchlorine dioxide to at least one organic acid is from 1:1 to 1:1000. 15.The method of claim 10 wherein said at least one organic is propionicacid or its salt, and a ratio of chlorine dioxide to at least oneorganic acid is from 1:1 to 1:1000.
 16. The method of claim 10 whereinat least one organic acid comprises citric acid or its salt.
 17. Themethod of claim 10 wherein the at least one organic acid is benzoic acidor its salt, and the ratio of chlorine dioxide to benzoic acid is from1:1 to 1:32,000, and the concentration of chlorine dioxide is from 1-50ppm in the aqueous system being treated.
 18. The method of claim 16wherein the ratio of chlorine dioxide to at least one organic acid isfrom 1:4 to 1:1000.